Project Summary Mesenchymal stem cells (MSC) in bone marrow provide regenerative capacity for bone, replacing and reinforcing the skeleton at load bearing sites. When we age or in prolonged bedrest, MSCs lose their regenerative potential, often measured by their proliferative and differentiation capacity. This loss of MSC health causes osteoporosis and delayed healing, ultimately resulting in decreased quality of life and increased medical costs. A fundamental knowledge gap preventing effective therapies in aging and MSC related regenerative medicine is how aging and bedrest impedes MSC health. The nucleus, central to all cellular activity, relies on both mechanical input as well as its molecular transducers to regulate intra-nuclear chromatin organization that ultimately determine cell function and fate. Thus, failure to transmit this information to the nucleus would lead to the breakdown of these processes. Here, we ask if aging is a process that limits information flow into the nucleus, ultimately diminishing its organizational capacity and responsiveness to outside stimuli. As we will show, disabling the mechanical connection between cytoskeleton and nucleus facilitated by Linker of Nucleoskeleton and Nucleoskeleton (LINC) complexes, impairs mechanosensitivity by affecting ?catenin and YAP/TAZ signaling. This leads to decreased proliferation and differentiation of mesenchymal stem cells. Our principal hypothesis is that loss of LINC-connectivity significantly contributes to MSC aging by disrupting nuclear mechanotransduction. Through this revision submitted in response to [PA16-442] - Changes in Cellular Architecture During Aging (R01), we will address our principal hypothesis through two specific aims, each using a distinct hypotheses to examine how inhibiting LINC complex function as well as how aging related loss of LINC complex limits MSC mechanosignaling of known mechanotransducers ?catenin and YAP/TAZ. We will further determine the force-induced mechanisms of how sustained physical activity protects LINC complex expression to augment MSC and bone mechanosignaling within the context of aging. If successful, we will establish, for the first time, a mechanistic understanding of how loss of LINC complex drives decreased mechanosensory capability in aging. Completion of these aims will provide research communities with (1) efficacy of LIV based regenerative modalities that improve LINC-mediated mechanosignaling and (2) foundational structure-function relationship data in healthy and aged stem cells.